Small noncoding RNAs are central components ofthe gene regulatory circuit in organisms. For proper health and development, organisms employ protein complexes containing small endogenous RNAs to control thousands of other genes through silencing mechanism. Genetics in model organisms implicate a link between small RNAs, Argonaute proteins and processes that regulate chromatin structure, suggesting that RNA interference ^RNAi) pathways can impact large domains of the genome. Our search for an analogous link in mammals directed us to purify from rat testes the piRNA Complex (piRC), which consists at least of mammalian Piwi and the Piwi-interacting RNAs. Fiwi-interacting RNAs (piRNAs) are a diverse class of small RNAs that are longer than microRNAs and derive from intergenic clusters of between 1-lOOkb long. Piwi, a subclade of the Argonaute family, is essential for fertility in flies and male mice, and has been genetically implicated in silencing of transposable elements. Despite these features, a rigorous understanding of piRC's molecular function is still lacking. This proposal seeks to dissect the molecular function ofthe piRNAs and piRC through these specific aims: 1) Functional analysis ofthe piRNA Complex;2) Regulation of piRNA cluster transcription;3) Understanding the Genesis of piRNAs. We will apply our current biochemical techniques to probe piRC's functional activity, and develop genetic and cell biology methodologies to understand the role of piRNA clusters. Piwi proteins have been shown to be essential for proper germ cell development in animals, including mammals. However, the mechanistic understanding and gene regulatory potential ofthis ribonucleoprotein is still uncharted. This research project aims to develop systems that will drive progress in a subject relevant to child health and human development.